RESEARC H Open Access Lack of association between stavudine exposure and lipoatrophy, dysglycaemia, hyperlactataemia and hypertriglyceridaemia: a prospective cross sectional study Phumla Z Sinxadi 1* , Jan-Stefan van der Walt 1 , Helen M McIlleron 1 , Motasim Badri 2 , Peter J Smith 1 , Joel A Dave 3 , Naomi S Levitt 3 , Gary Maartens 1 Abstract Background: Stavudine continues to be widely used in resource poor settings despite its toxicity. Our objective was to determine association between plasma sta vudine concentrations and lipoatrophy, concentration s of glucose, lactate and triglycerides. Methods: Participants were enrolled in a cross-sectional study with lipoatrophy assessment, oral glucose tolerance test, fasting triglycerides, finger prick lactate, and stavudine concentrations. Individual predictions of the area under the concentration curve (AUC) were obtai ned using a population pharmacokinetic approach. Logistic regression models were fitted to assess the association between stavudine geometric mean ratio > 1 and impaired fasting glucose, impaired gluco se tolerance, hyperlactataemia, hypertriglyceridaemia, and lipoatrophy. Results: There were 47 study participants with a median age of 34 years and 83% were women. The median body mass index and waist:hip ratio was 24.5 kg/m 2 and 0.85 respectively. The median duration on stavudine treatment was 14.5 months. The prevalence of lipoatrophy, impaired fasting glucose, impaired glucose tolerance, hyperlactataemia, and hypertriglyceridaemia were 34%, 19%, 4%, 32%, and 23% respectively. Estimated median (interquartile range) stavudine AUC was 2191 (1957 to 2712) ng*h/mL. Twenty two participants had stavudine geometric mean ratio >1. Univariate logistic regr ession analysis showed no association between stavudine geometric mean ratio >1 and impaired fasting glucose (odds ratio (OR) 2.00, 95% CI 0.44 to 9.19), impaired glucose tolerance (OR 1.14, 95% CI 0.07 to 19.42), hyperlactataemia (OR 2.19, 95%CI 0.63 to 7.66), hypertriglyceridaemia (OR 1.75, 95%CI 0.44 to 7.04), and lipoatrophy (OR 0.83, 95% CI 0.25 to 2.79). Conclusions: There was a high prevalence of metabolic complications of stavudine, but these were not associated with plasma stavudine concentrations. Until there is universal access to safer antiretroviral drugs, there is a need for further studies examining the pathogenesis of stavudine-associated toxicities. Introduction Stavudine is no longer recommended as part of first line combination antiretroviral therapy (ART) because of a high cumulative risk of toxicity, notably symptomatic hyperlactataemia/lactic acidosis, lipoatrophy, and periph- eral neuropathy [1,2]. In addition, stavudine ca uses dyslipidaemia a nd insulin resistance, and is an indepen- dent risk factor for the development of new onset dia- betes mellitus [3]. Although the World Health Organization (WHO) ART guidelines for resource-lim- ited settings urge countries “ to begin planning to move away from stavudine-containing regimens” [4], stavudine continues to be widely used in standardised first-line regimens in low- and middle-income coun tries as it has a low acquisition cost, is available in fixed dose * Correspondence: phumla.sinxadi@uct.ac.za 1 Department of Medicine, Division of Clinical Pharmacology, University of Cape Town, K45 Old Main Building, Groote Schuur Hospital, Observatory, 7925, Cape Town, South Africa Sinxadi et al. AIDS Research and Therapy 2010, 7:23 http://www.aidsrestherapy.com/content/7/1/23 © 2010 Sinxadi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly ci ted. combination formula tions and does not require labora- tory monitoring for toxicity. In 2006, WHO recommended reduced doses of stavu- dine following the findings of a systematic review that lower doses caused less toxicity without reducing effi- cacy [1,5]. Most, if not all, of stavudine’s adverse dr ug reactions are thought to be mediated by mitochondrial toxicity and to be dose related [6,7]. Therefore it is rea- sonable to assume that higher plasma concentrations of stavudine mig ht be associated with more toxicity. How- ever, there are “ no clear plasma concentration-effect relationships” with nucleoside reverse transcriptase inhi- bitors like stavudine, which are pro-drugs that require intracellular tri-phosphorylation for antiviral activity [8]. A retrospective study from the Netherlands reported a correlation between lipoatrophy and higher stavudine plasma concentrations [9], but data correlating stavudine plasma concentrations with other metabolic adverse drug reactions are lacking. We investigated whether there was an association between stavudine plasma concentrations and lipoatro- phy or concentrations of glucose, lactate and triglyceride in a population where stavudine use is likely to be wide- spread in the medium term: African HIV-infected adults. Methods Study design and participants We conducted a prospective cross sectional study between February 2007 and January 2008. Ambulatory HIV-infected African black adults who presented for a routine follow up visit at public sector antiretroviral clinics in Cape Town were recruited by convenient sampling. Participants were eligible i f they were on sta- vudine-based therapy for a minimum of 6 months. Par- ticipants with renal or hepatic disease, active opportunistic infections, known diabetes or dyslipidae- mia, or self-reported non-adherence were excluded. All participants gave informed consent. The University of Cape Town research ethics committee approved the study. Clinical and laboratory evaluations Participants fasted overnight and underwent an oral glu- cose tolerance test (OGTT). Impaired fasting glucose (IFG), impaired glucose tolerance (IGT) and diabetes were defined according to the American Diabetes Asso- ciation criteria [10]. Fasting triglycerides were deter- mined at 0 min of the OGTT. Hypertriglyceridaemia was defined according to the NCEP III criteria [11]. Fin- ger prick lactate was measured before the glucose load- ing using the Accutrend® lactate meter (Roche, Basel, Switzerland). Hyperlactataemia was defined as a lacta te concentration greater or equal to 2.5 mmol/L. Lipoatrophy was determined by self-reported periph- eral fat loss using a validated questionnaire [12]. Lipoa- trophy was rated as absent (score = 0), mild (noticeable on close inspection, score = 1), moderate (readily noticeable by participant, score = 2) or severe (readily noticeable to a casual observer, score = 3) in each of four areas (face, arms, legs and buttocks). The lipoatro- phy score c ould range from 0 to 12. Lipoatrophy was regarded to be present if the score was 1 or above. Self reported adherence was determined using a stan- dard 4-day adherence questionnaire administered by trained field workers [13]. We reviewed medical records to determine duration on antiretroviral therapy and cur- rent CD4+ lymphocyte counts and viral load. Current CD4+ count was regarded as the count measured within 3 months of the study visit. We measured plasma stavudine concentrations at 0, 30, and 120 minutes of the OGTT. We collected the blood samples using heparinised tubes that were imme- diately placed on ice until centrifugation within 4 hours, and then kept in a minus 80°C freezer until analysis. Stavudine was assayed by liquid chromatography tan- dem mass s pectrometry using a validated method on an API 4000 mass spectrometer. The mobile phase con- sisted of gradient of acetonitrile and 0.5% glacial acetic acid. Chromatography was performed on a Phenomonex Synergi f usion C18 column maintained at 25°C. Reser- pine was used as an internal standard. 50 μL of each sample was precipitate d with acetonitrile containing the internal standard, centrifuged and 5 μLofthesuperna- tant injected onto the column. Standard curves in the range 0.02 - 6 μg/mL and appropriate quality control samples were run with each batch. The lower limit of quantification was 20 ng/mL. Inter- and intra-day coeffi- cients of variatio n were below 9% for all quality control samples. Pharmacokinetic analysis The aim of the pharmacokinetic analysis was to obtain a prediction of each participant’s apparent stavudine clear- ance (CL/F) for calculation of the area under the con- centration curve (AUC), where AUC = dose (ng)/ clearance (L/h). The data were analysed using nonlinear mixed effects modelling with NONMEM® (version VI level 2.0; ICON Development Solutions, Ellicott City, MD, USA). Given the sparse data (1-3 observations per participant), a model developed using rich stavudine concentration data from a sep arate study our group has conducted of African adults from the same community was used [14]. The population pharm acokinetic para- meter estimates were fixed to: apparent clearance 17.8 L/h/70 kg 3/4 (between subject variability 17%CV), appar- ent volume of distribution 33.5 L/kg, first-order absor p- tion rate constant 11.1/h (between subject variability Sinxadi et al. AIDS Research and Therapy 2010, 7:23 http://www.aidsrestherapy.com/content/7/1/23 Page 2 of 6 125%CV), absorption lag time 0.41 h, proportional resi- dual variability 27% and additive residual variability 10 ng/mL. Individual pharmacokinetic parameter sets were then obtained using Bayesian estimation given these model parameters and the observed data. The geometric mean ratio (GMR) was calculated by comparing the individual log-transformed AUC to the mean log-trans- formed AUC of the overall population. Statistical analysis Means (standard deviation (S.D)) and medians (inter- quartile range) were used to descr ibe parametric data and non-parametric data, respectively. Categorical data were compared using c 2 test (or Fisher’s exact test), and continuous data were compared using student’sT-test or Mann-Whitney test, whichever was appropriate. Logistic regression models were fitted to assess the asso- ciation between GMR > 1 and IFG, IGT, hyperlactatae- mia, hypertriglyceridaemia and lipoatrophy. Linear regression models were fitted to assess the association between log-transformed stavudine area under the curve and the following variables: concentrations of glucose, lactate and triglycerides, and lipoatrophy scores. All tests were two-sided, and a P-value < 0.05 was consid- ered significant. Analyses were performed using SPSS (version 17, SPSS Inc, Chicago, Illinois, USA) Results Forty seven black participants were included for the ana- lysis. Median (IQR) age was 34 (30-38) years. Thirty nine part icipants were female. Median (IQR) weight and body mass index were, 61.0 (54.4 to 73.8) kg and 24.5 (21.5 to 30.4) kg/m 2 , respectively. Median waist to hip ratio was 0.85 (0.80 to 0.92). Median (IQR) current CD4 count was 304 (234-516) cells/μL. Twelve participants were virologi- cally suppressed, 6 had viral load above 50 copies/mL and 29 had no viral load data. Forty and seven participants were on 30 mg and 40 mg of stavudine, respectively. Twenty six, twenty and one participants were on efavir- enz, nevirapine and lopinavir, respectively. All partici- pants were on lamivudine. The median (interquartile range (IQR)) fasting glucose concentration was 4.9 (4.7 to 5.4) mmol/L and the mean (standard deviation (sd)) 2 hour glucose concentration was 5.34 (1.43) mmol/L. Nine and two participants had IFG and IGT, respectively. The mean (sd) lactate concentration was 2.26 (0.78) mmol/L and 15 participants had hyperlactataemia. The median (IQR) triglyceride concentration was 1.17 (0.85 to 1.60) mmol/L and 11 participants had hypertriglyceri- daemia. The median (IQR) lipoatrophy score was 0 (0 to 9) and 16 patients had lipoatrophy. A total of 122 st avudine concentrati ons from 47 parti- cipants were analysed. Eleven participants had no pre- dose concentrations because they took their stavudine morni ng doses prior to the OGTT. The ob served stavu- dine concentrations plotted again st the model predic- tionsareshowninFigure1.Stavudineexposurewas expressed with the calculated AUC. The median (inter- quartile range) stavudine AUC was 2191 (1957 to 2712) ng*h/mL. The mean (standard deviation) log-tran s- formed AUC was 3.36 ± 0.10 ng*h/mL. 22 p articipants had a GMR greater than 1. Figure 1 Plasma stavudine concentrations vs time after dose, collected during OGTT in 47 HIV-1 infected participants.OGTT=oral glucose tolerance test, d4T = stavudine. The solid line indicates the median predicted concentrations and the dashed lines the 90% prediction interval of a model developed using rich stavudine concentration-time data. The lower limit of quantification (20 ng/mL) is shown by a dotted line. Open circles indicate stavudine concentrations when stavudine was taken before the OGTT, and the solid diamonds are stavudine concentrations collected when the stavudine dose was taken during the OGTT (i.e. the 0-hour OGTT was collected pre-dose). Sinxadi et al. AIDS Research and Therapy 2010, 7:23 http://www.aidsrestherapy.com/content/7/1/23 Page 3 of 6 We found no association between log-transformed sta- vudine AUC and metabolic parameters expressed as continuous variables (Table 1). We also showed no asso- ciation between stavudine geometric mean ratio >1 and abnormal metabolic pa rameters expressed as categorical variables (Table 2). We found an association between duration and triglycerides concentrations (beta coef fi- cient 95%CI = 0.02 (0.01 to 0.04) p = 0.004. No signifi- cant association was found between duration and glucose and lactate concentrations as well as lipoatrophy scores. Discussion Despite guidelines recommending that the use of stavu- dine be avoided because of its toxicity, it continues to play a critical role in scaling up antiret roviral therapy in resource poor settings. Therefore, studies examining pathogenesis of stavudine toxicity are still relevant. We found no association between stavudine AUC and the lipoatrophy scores or concentrations of glucose, lactate and triglycerides. To our knowledge this is the first study to evaluate the association betw een plasma stavu- dine c oncentrations and serum glucose, lactate and tri- glycerides. We found high prevalence of metabolic abnormalities in this black African cohort with a median duration of stavudine exposure of 14.5 months: lipoatro- phy (34%), dysglycaemia (23%), hyperla ctataemia (32%), and hypertriglyceridaemia (23%). We found an associa- tion between duration and triglycerides concentrations. A meta-analysis from randomised control trials and cohort studies showed that switching from higher to lower doses of stavudine, or startin g at lower doses, is associated with improvement in stavudine toxicity with- out loss of efficacy [5]. Switching to lower doses of sta- vudine was associated with decreased drug exposure, mitochondrial DNA repletion, partial reversal of lipoa- trophy, improvement in lactate and lipids [5, 15,16]. Therefore, stavudine toxicity is dose related. The lack of an association between stavudine AUC and all the meta- bolic abnormalities that we found can be explained as follows: First, like all nucle oside/nucleotide reverse tran- scriptase inhibitors (NRTIs), stavudine is a p ro-drug that must be converted intracellularly into its tripho- sphate moiety (d4T-TP) to exert antiviral activity by competing with endogenous nucleotides to terminate HIV replication [17]. The d4T-TP also inhibits mito- chondrial DNA polymer ase gamma in a dose dependent manner in cells of various tissues, and effectively termi- nates mitochondrial replicati on with subsequent mito- chondrial damage or depletion: the common pathway for stavudine related toxicity. Intracellular triphosphate conc entrations, but not NRTI parent drugs, have gener- ally been shown to have a good correlation with anti- viral activity [17-21], as well as toxicity [22]. The cellular processes that affect the relationship between plasma NR TI and intracellular triphosphate concentra- tions include variation in expression of drug transpor- ters, rate limiting steps or saturated phosphorylation steps, cell activation state, and drug interactions [17,19]. Second, clinical manifestations of stavudine toxicity are also influenced by host susceptibility such as age, sex, advanced HIV disease and genetic susceptibility [17,23-26]. Third, it seems that stavudine toxicity is cumulative, as it is shown to be dose related and is asso- ciated with pr olonged duration on treatment [5,9,15,16]. Therefore, differences in plasma concentrations, if they exist, are likely to b e small, and a very large study will be needed to detect the difference. Although stavudine related toxicity is well documen- ted, to date, few studies have investigated pharmacoki- netic relationship with stavudine toxicity. Our findings are different to a case-control study conducted by ter Hofstede et al, which reported that cases with lipoatro- phy had higher stavudine exposure than controls [9]. However, there were no statistically significant differ- ences in geometric means of concentration ratios between the cases and cont rols. The discrepancy between their findings and ours could possibly be explained by differences in study design and participant selection. Ter Hofstede et al conducted a retrospective study. Exposure was represented by a time-adjusted con- centration ratios derived from a historic population. In contrast, our study was conducted prospectively and we used stavudine AUC derived from individual clearances obtained from a pharmacokinetic model of intensively sampled participants from the same community [14]. The estimated AUC of 2191 ng*h/mL in our population Table 1 Univariate linear regression analysis of stavudine log-transformed AUC and metabolic parameters Variable Beta coefficient (95% CI) p-value Fasting glucose -0.02 (-0.06 to 0.04) 0.54 2 hour glucose -0.02 (-0.04 to 0.00) 0.09 Lactate 0.00 (-0.04 to 0.04) 0.91 Triglycerides 0.00 (-0.05 to 0.05) 1.00 Lipoatrophy score -0.00 (-0.01 to 0.01) 0.37 Table 2 Univariate logistic regression analysis of stavudine geometric mean ratio >1 and metabolic parameters Variable Odds ratio (95%CI) p-value Impaired fasting glucose 2.00 (0.44 to 9.19) 0.37 Impaired glucose tolerance 1.14 (0.07 to 19.42) 0.93 Hyperlactataemia 2.19 (0.63 to 7.66) 0.22 Hypertriglyceridaemia 1.75 (0.44 to 7.04) 0.43 Lipoatrophy score >0 0.83 (0.25 to 2.79) 0.83 Sinxadi et al. AIDS Research and Therapy 2010, 7:23 http://www.aidsrestherapy.com/content/7/1/23 Page 4 of 6 is similar to that found in the richly sampled South Afri- canpopulationweusedforthepopulationmodel[14] and to contr ol patients from the US [27], but is higher than reported in Indian patients [28] or the Summary of Product Characteristics of Zerit [29]. It is possible that stavudine exposure is high in our population, which may account for the high prevalence o f metabolic abnormalities we observ ed. However, the prevalence of metabolic abnormalities on stavudine-containing regi- mens that we found were comparable to other published studies with variable duration of follow-up and different additional antiretroviral drugs: lipoatrophy (20-42%) [30-33], dysglycaemia (3-25%) [34], hyperlactataemia (15-35%) [35], and hypertriglyceridaemia (22-71%) [36,37]. We found an association between duration and triglycerides concentrations and this has been reported before [36]. Our study had a few limitations. First, we measured stavudine concentrations in plasma and not the active intracellular triphosphorylated metabolite. Second, we used sparse sampling instead of intensive sampling. However, a population approach allowed us to predict individual AUCs, an acceptable measure o f drug expo- sure. Third, we did not have data on genetic polymorph- isms. Fourth, sample size of this study was small, and therefore might have insufficient power to detect rela- tively small effects of pla sma concentrations on meta- bolic abnormalities. However, this sam ple size is larger than in other pharmacokinetic studies that have exam- ined the association between stavudine concentrations and metabolic toxicity [9,22]. Future studies examining the pathogenesis of stavu- dine-associated toxicities should have adequate power and preferably be longitudinal. Relevant genetic studies should also be done in the populations where stavudine will still be used in the medium term. Physiologically based pharmacokinetic models that take into account the temporal fluctuations and intracellular cascade steps of plasma NRTIs and metabolites should be used to estab- lish pharmacokinetic-pharmacodynamic relationships. In conclusion, we di d not find an association between stavudine exposure and metabolic complications. Despite guidelines recommending that the use of stavu- dine be avoided b ecause of its toxicity, it is still widely used in resource poor settings. Until there is universal access to safer drugs, there is a need for further studies examining the pathogenesis of stavudine-associated toxicities. Acknowledgements The authors are grateful to Ms Carmen Delport (study coordinator) and her team, for the help with collecting blood samples; Ms Alicia Evans and pharmacology laboratory team for the sample preparation and analysis; and to the patients for their participation in this study. This study was funded by the World Diabetes Foundation, South African Department of Health and the South African Medical Research Council. The funding bodies had no role in study design; in collection, analysis and interpretation of the data; in writing of the manuscript; and in the decision to submit the manuscript for publication. Author details 1 Department of Medicine, Division of Clinical Pharmacology, University of Cape Town, K45 Old Main Building, Groote Schuur Hospital, Observatory, 7925, Cape Town, South Africa. 2 Department of Medicine, Clinical Research Support Unit, University of Cape Town. J45-46 Old Main Building, Groote Schuur Hospital, Observatory, 7925, Cape Town, South Africa. 3 Department of Medicine, Division of Diabetic Medicine and Endocrinology, University of Cape Town. J47 Old Main Building, Groote Schuur Hospital, Observatory, 7925, Cape Town, South Africa. Authors’ contributions PZS participated in the study design, acquisition of data, data analysis and interpretation, and drafted the manuscript. JSvdW participated in study design, population pharmacokinetic analysis and helped to draft and critically revise manuscript. HMM participated in study design, data interpretation, and critical revision of the manuscript. MB performed statistical analysis and helped to draft and revise manuscript. PJS performed analysis of the samples and helped to draft the manuscript. JAD participated in study design, acquisition of data and critically revised the manuscript. NSL participated in study design and acquisition of data. GM conceived of the study, participated in study design, data interpretation, and critically revised manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Received: 22 March 2010 Accepted: 14 July 2010 Published: 14 July 2010 References 1. 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Domingos H, da Cunha , Paniago AMM, Martins DM, Elkhoury EB, de Souza AS: Metabolic effects to the Highly Active Antiretroviral Therapy (HAART) in AIDS patients. The Brazilian Journal of Infectious Diseases 2009, 13:130-136. doi:10.1186/1742-6405-7-23 Cite this article as: Sinxadi et al.: Lack of association between stavudine exposure and lipoatrophy, dysglycaemia, hyperlactataemia and hypertriglyceridaemia: a prospective cross sectional study. AIDS Research and Therapy 2010 7:23. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Sinxadi et al. AIDS Research and Therapy 2010, 7:23 http://www.aidsrestherapy.com/content/7/1/23 Page 6 of 6 . this article as: Sinxadi et al.: Lack of association between stavudine exposure and lipoatrophy, dysglycaemia, hyperlactataemia and hypertriglyceridaemia: a prospective cross sectional study. AIDS. RESEARC H Open Access Lack of association between stavudine exposure and lipoatrophy, dysglycaemia, hyperlactataemia and hypertriglyceridaemia: a prospective cross sectional study Phumla Z Sinxadi 1* ,. as part of first line combination antiretroviral therapy (ART) because of a high cumulative risk of toxicity, notably symptomatic hyperlactataemia/ lactic acidosis, lipoatrophy, and periph- eral